Long-term goal: The long-term goal of this renewal is to unravel the molecular mechanism(s) underlying nitration/nitrosation/oxidation reactions initiated by pro-inflammatory reactive nitrogen species (RNS) in biological systems. Previous research demonstrated that hydrophobic membranes enhance nitration and oxidation of intramembrane tyrosyl peptides. Hypothesis: We hypothesize that the hydrophobic microenvironment enhances nitration, nitrosation, and oxidation reactions, induced by RNS, in membrane-associated proteins. Specific aims: First, we will investigate the microenvironmental factors (hydrophobicity, membrane proximity, bicarbonate/carbon dioxide concentration, membrane oxygen levels, amino acid sequence) influencing tyrosine nitration/oxidation and lipid nitration/oxidation in the membrane. Next, we will determine the effect of nitric oxide on lipid nitration and transmembrane nitration of tyrosyl probes. The objective here is to monitor the transition from nitration to nitrosation reactions in membranes, and finally we will evaluate the influence of membrane versus cytosolic protein association on the nitration/oxidation/nitrosation profiles induced by pro-inflammatory nitrating species. Methods: Initially, we will use ?-synuclein, a 19 kDa tyrosine-containing protein, the green fluorescence proteins (GFP), and red blood cells. Both ?-synuclein and GFP can be readily expressed in cytosolic and membrane-associated forms. New intramembrane nitration and nitrosation probes will be synthesized and products and intermediates identified by HPLC, LC/MS, ESR, and chemiluminescence techniques. Significance: Protein nitrotyrosine and nitrated lipids are diagnostic markers of RNS (peroxynitrite, nitrogen dioxide radical) and mediators of the pathological development of inflammatory diseases. Novelty: This proposal will provide new insights on the role of hydrophobic membranes on nitration/oxidation/ nitrosation of membrane-associated proteins. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]